透明颤菌血红蛋白基因的植物化改造合成及其在大肠杆菌中功能的鉴定

 根据透明颤菌血红蛋白（VHb）的氨基酸序列，按照植物偏爱密码子，对透明颤菌血红蛋白基因（vgb）进行优化改造，同时考虑到可实现分别克隆拼接的酶切位点，设计并合成了22条单链DNA短片段，通过拼接获得了全长450 bp的透明颤菌血红蛋白基因vgbM，并将其克隆至pUC19上，获得重组质粒pGSVHB。利用设计的引物，通过PCR在vgbM基因的5′端引入NcoⅠ位点，将其克隆到原核生物高效表达载体pBV221上，获得质粒pBV221SVHB。转化E.coli. BL21并经热激诱导表达后，在培养物中检测到了约16 kD的VHb蛋白。经CO差示光谱分析测定，该蛋白具有生物活性。为透明颤菌血红蛋白基因vgbM在植物基因工程研究中的应用奠定了基础。

The Synthesis of the vgbM Gene with Plant Preferential Codon and Its Function Identification in E.coli

According to the amino acid sequence of Vitreoscilla hemoglobin (VHb), a novel gene encoding the VHb protein was designed with plant preferential codon. First 22 oligodeoxynucleotide fragments were synthesized based on the design, meanwhile, certain restriction enzyme sites for further cloning were considered. Three small DNA fragments, which were formed by annealing certain oligonucleotide fragments, were sub-cloned respectively. Then the vgbM gene, which is 450 bp in length, was cloned into pUC19 by ligating the three small DNA fragments. The recombinant plasmid was named pGSVHB. The pBV221SVHB was further constructed by inserting the vgbM gene into the plasmid pBV221 after a Nco I site adding at 5'-terminal through PCR. Subsequently, the vector was introduced into E. coli, BL21 strain. The 16 kD VHb protein was detected after hot-shock inducing expression of the engineered E. coli. The biological activity of the expressed VHb was demonstrated by CO difference spectrum analysis. This work has laid a foundation for further studies to transfer vgbM gene into plants.